Plasma membrane gap junctions interconnect cytoplasms of adjacent cells and permit transfer of ions and low molecular mass metabolites. They have been postulated to play critical, but as yet, incompletely defined roles in the control of cell growth. The hypothesis that loss of junctional communication results in loss of growth control is of particular importance to the cancer problem. Development of tumors in vivo that eventually become invasive and metastatic malignancies could result from this lack of intercellular communication. The Rous sarcoma virus, containing the src oncogene encoding pp60v-src, may also induce the neoplastic transformed cell phenotype by abrogation of junctional communication. The primary objective of this research is to investigate the molecular mechanisms by which tyrosine (tyr) phosphorylation of the gap junction protein, connexin43, changes junctional communication in RSV-transformed fibroblasts. Specific Aim 1 will determine if phosphorylation of connexin43 on tyr is correlated with loss of channel function in RSV- infected cells. Research will compare alterations in junctional communication to connexin43 phosphorylation on serine (Pser) and tyrosine (Ptyr) amino acids in fibroblasts containing pp60src mutants that are temperature sensitive for transformation, or altered in their membrane binding or modulatory SH2 domains, and RSV-transformed cells treated with agents such as cAMP, retinoic acid, and genistein that reverse the transformed state. Specific Aim 2 will determine if pp60c-src disrupts junctional communication through phosphorylation of connexin43 on tyr in mitotic-phase fibroblasts and cells transformed by polyomavirus which contain kinase-activated pp60c-src. Experiments for these aims will use immunoprecipitation, SDS-PAGE, phosphoamino acid and 2-D tryptic peptide analyses, immunoblotting, kinase reactions, and microinjection techniques. Specific Aim 3 will identify connexin43 Pser and Ptyr sites and determine if mutations in these amino acids affect channel function or are required for pp60v-src disruption of junctional communication. Phosphorylation sites will be identified by immunoprecipitation of phosphorylated connexin43 protease fragments and in vitro kinase labeling. Phosphorylation site mutants will be generated by directed mutagenesis in cloned connexin43 genes, and functional mutants will be assayed by transfection and expression of these mutants in non-communicating cells.